Age-related macular degeneration (AMD) is a major cause of retinal damage, the leading cause of blindness in Western countries and, as the name implies, a disease of aging. Like other age-related diseases, AMD is particularly challenging to study because it takes decades to develop and so any research model must recapitulate the conditions of an older organism. Genome-wide association studies (GWASs) and linkage analyses have provided the first clues to what might cause AMD. These studies identified three single nucleotide polymorphisms (SNPs) that are strong risk factors for AMD.1 One SNP lies in the 402H allele in the CFH gene and the two others are tightly linked and lie in the neighboring HTRA1 and ARMS2 genes. These SNPs confer the most significant genetic risk factors in the history of GWAS studies in human genetics. People homozygous for these SNPs have a 50-fold increased risk of AMD. How these mutations might cause sight to deteriorate is unclear, however, because the underlying molecular mechanisms of AMD are unknown. Recently, however, our unbiased proteome analysis suggested super oxide dismutase (SOD) mechanisms are perturbed in affected cells and that, over time, this introduces reactive oxidative species (ROS) mediated cellular insults that eventually manifest as AMD. If ROS metabolism is indeed disrupted in AMD, then we might finally begin to address the causes of the disease. We believe the hurdles faced in finding causes and treatments for AMD could be circumvented by stem cell technologies. To this end we have found a way to differentiate stem cells from patients into retinal cells. Moreover, we developed a protocol that recapitulates aging in these patient-stem-cell-derived retinal cells. Finally, through gene-targeting technology, we can manipulate the stem cell genome, targeting disease- associated SNPs, to determine the individual contributions of each. By applying these powerful methodologies, we believe we can finally identify the root causes of AMD and so begin to develop new therapies. Our goals will be accomplished in two specific aims: Aim 1A. Use the CRISPR/Cas9 system to convert HTRA1 and ARMS2 alleles from low-risk to high risk in patient-derived stem cells. Aim 1B. Determine the individual contribution of human HTRA1 and ARMS2 alleles to AMD pathogenesis. Test whether CRISPR conversion from low to high-risk AMD alleles in Aim 1A affects ROS levels in cells. Aim 2. Test the function of patient-stem-cell-derived RPE in a human-mouse chimera, in vivo assay.